The formation of myosin-lI filaments is fundamental to contractile and motile processes. Understanding how filament assembly and disassembly are controlled is essential for determining how myosin-II rapidly responds to changing conditions within the cell (e.g. during cell division or in response to a chemotactic signal). Several proteins have been shown to stabilize filamentous myosin-II assemblies; however mts1, a member of the Ca2+regulated S100 family of proteins, is the first protein to be identified that promotes the monomeric, unassembled state. In addition to its involvement in generalized cell motility, mts1 is directly implicated in the mesenchymal-epithelial transition and is a major metastatic factor. Thus mts1 is an excellent target for investigating the molecular mechanisms controlling the localized assembly/disassembly of myosin-II that are relevant to motility, development and metastasis. Biochemical studies will be used to establish the molecular basis by which mts1 regulates the monomer-polymer equilibrium of myosin-II and how myosin-II heavy chain phosphorylation regulates mts1 activity. High resolution structural studies of mts1 bound to the myosin-II heavy chain will allow a detailed atomic description of the mts1/myosin-II interaction, provide constraints for the biochemical mechanism by which mts1 regulates myosin-II function and provide specific information that will assist in biosensor construction and mutagenesis. Using environmentally sensitive fluorophores that are sensitive to solvent polarity, we will develop novel mts1 biosensors that report calcium or target binding for use in individual living cells. These unique reagents will allow the temporal and spatial distribution of activated mts1 to be examined directly during the motility cycle, and will permit a direct correlation between localized, transient activation of mts1 and the regulation of specific myosin-II assemblies during directed motility. Altogether, this information will be leveraged to establish a complete biochemical model for mts1-mediated regulation of myosin-II function during cellular motility.